Amplification of nucleic acids and analysis of the resulting amplification products has revolutionized the basic and clinical sciences. Applications of these techniques include molecular cloning, nucleic acid sequencing, genotyping, detection and identification of single nucleotide polymorphisms (SNP) and other polymorphisms and mutations and the quantitation of gene expression,
Various techniques for nucleic acid amplification have been developed, such as strand displacement amplification, transcription-based amplification, and polymerase chain reaction (PCR).
Use of PCR in large scale research projects and in clinical applications entails amplification of many distinct target sequences with the concomitant generation of a great number of PCR amplicons. As the scale of such projects increases, it has become cost prohibitive and inefficient to undertake the necessary reactions singly. Thus, there is great interest in developing methods of performing multiple amplification reactions in parallel in the same reaction vessel using a common pool of template and reagents.
Such multiplex PCR methods, in which multiple pairs of target-specific primers are used to co-amplify multiple targets, have met with only qualified success. Combining all the required primers in the same tube greatly increases the frequency of formation of primer-dimer and other spurious amplification products. As the number of primer pairs rises in multiplex PCR, the number of potential primer-dimer interactions (or spurious amplicons generated by two different primers) increases exponentially according to the number of primers used.
Even with careful attention paid to the design of the multiplex primer pairs to avoid obvious primer-dimer incompatibilities, conventional multiplex PCR is generally limited to about 10-20 simultaneous amplification reactions before undesired amplification products predominate. Different approaches to ameliorate the problems associated with multiplex PCR have been developed, but none with unqualified success.
PCT publication WO 96/41012 discloses a method for multiplex PCR that entails two rounds of amplification and that uses primer pairs comprising template-specific sequences at their respective 3′ ends and universal, or common, primer sequences at their respective 5′ ends. The first round of amplification uses the specific primer sequences and the second amplification uses the universal primer sequences. The second round normalizes differential binding of the specific primers to different templates.
Another multiplex method uses a single specific primer for each target and a single common primer. N. E. Broude, et al., Proc. Natl. Acad. Sci. USA 98:206-211 (2001). This method still suffers from the amplification of spurious products, however, and therefore remains limited in its application.
Yet another multiplex method uses a precircle probe to form a hybridization complex with sequence of interest. After hybridization complex is contacted with a ligase to form a closed circular probe, and cleaving the dosed circular probe at the cleavage site to form a cleaved probe. In the precicle probe the common sequences are in opposite directions, but in the cleaved probe they are facing toward each other and can be used for amplification by PCR. U.S. Pat. No. 6,858,412.
The oligonucleotide ligation assay (OLA; sometimes referred to as the ligation chain reaction (LCR)) involves the ligation of at least two smaller probes into a single long probe, using the target sequence as the template for the ligase. See generally U.S. Pat. Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; and WO 89/09835, all of which are incorporated by reference.
Many of the methods are used to archive the highest level of the enrichment at the early enzymatic steps and use simple detection steps after selective amplification. Thus, remains a need in the art for methods of simultaneous multiplex amplification of large numbers of specific nucleic acid sequences that relatively simple, cost effective and do not require highest degree of enrichment.
Other type methods rely on the converting the small RNA into cDNA by using short LNA primers or attaching primer at the 3′ end of the RNA molecule and using it as template for reverse transcription.
For other type of RNA analysis like RNA expression profiling the quality of the RNA is important issue. Often the degradation of RNA or chemically modifications of RNA in FFPE samples prevent usage of those samples in expression analysis. The conversion of the RNA molecule into cDNA molecule is mostly affected by the RNA quality. Developing new method which avoiding cDNA synthesis would be beneficial for such RNA sample usage.
In each of these methods, analysis of the small RNAs or damaged RNA is problematic. Accordingly, it is an object of the invention to provide compositions and methods for the detection and quantification of the products by capturing the ribonucleotides on the template and converting the captured molecule into the amplifiable detectable decoding sequence.